In recent years, a demand for the analysis of nucleic acid base sequences has been considerably increasing for the purpose of detecting causative genes of genetic diseases, evaluating effectiveness or side effect of drugs, detecting genetic mutation involved in cancerous diseases, or the like. Therefore, information obtained from the results of analysis needs to have high precision.
As a method of reading a base sequence of a nucleic acid molecule with high precision, for example, there is proposed a method of determining a consensus sequence of nucleotides in a template nucleic acid segment, the method including: providing a sense strand and an antisense strand of the template nucleic acid segment in a contiguous nucleic acid molecule; and sequencing both the sense strand and the antisense strand in a polymerase mediated, template dependent sequencing process (PTL 1). In PTL 1, it is described that “By virtue of the circular nature of the template structure, repeated sequencing of the same molecule can be performed in plural times. In other words, the sequencing process progresses around a completely contiguous sequence, and each segment from the complementary sequences is repeatedly sequenced so that sequence data of the segment and sequence data in each segment can be repeatedly obtained. All or portions of such sequence data are then useful in deriving a consensus sequence for the template and its various segments” (Paragraph [0055]).
In addition, PTL 2 discloses adapters that include a double-stranded nucleic acid region and are usable for generating single-stranded constructs of nucleic acid for sequencing purpose. In PTL 2, it is described that “This ensures that, when the construct is sequenced, each position in the double-stranded nucleic acid is not merely observed once, but is in fact interrogated twice. This gives greater certainty that each position in the nucleic acid has been observed and that the aggregate call for both bases at each position is of a greater quality score than would be possible with a single observation” (Paragraph [0038]). It is further described that “The ability to interrogate each position twice is also helpful for differentiating between methylcytosine and thymine using stochastic sensing. These two bases result in very similar current traces when they pass through and interact with a transmembrane pore. It can therefore be difficult to differentiate between the two. However, interrogation of each position in a nucleic acid twice will allow such differentiation because the complementary base for methylcytosine is guanine, whereas the complementary base for thymine is adenine. Methylcytosine has of course been linked with various diseases, including cancer” (Paragraph [0042]).